Study On The Role Of Integrin αvβ6 In Gastric And Colon Cancers

Background:As an adhesion molecules that only expresses in epithelial tissues that have undergone malignant transformation,integrin avβ6 has been studied in colon cancer,breast cancer,pancreas cancer and oral cancer,and significant achievements have been achieved, especially in colon cancer.These studies comfirmed that integrinαvβ6 had an important role in promoting generation,progression,infiltration and metastasis of the tumours above. Surprisingly however,little information about integrinαvβ6 in gastric carcinoma has been reported.Gastric carcinoma is a frequent malignant tumour with poor prognosis in human.In china,the incidence rate and death rate of gastric carcinoma is the highest of all the malignant tumours,and in the world,it has become the second cause of death.Colon cancer also occupies a very important part of tumour death.Thus,it has been an imminent task for the medical workers to search an efficiently means to cure gastric carcinoma and colon cancer.Since integrinαvβ6 has an important role in promoting the progression of the tumours of colon,breast,pancreas and oral,if is it similar in gastric carcinoma,and if could it be an efficiently target for curing gastric carcinoma and colon cancer? Our study is based on these background and purpose.Part One The role ofintegrinαvβ6 in the progression of gastrci carcinoma cellsObjective To study the roles of integrinαvβ6 in the survival,proliferation,infiltration and metastasis of gastric carcinoma cells.Methods Firstly,flow cytometry was used to screening the gastric carcinoma cell(AGS) that expressing integrinαvβ6.Then,we constructed the small interfering RNA (siRNA)targetting integrinαvβ6,and transfected the siRNA into AGS cells with liposome -2000.RT-PCR and Western blotting were used to detect the inhibiting effects of the siRNA on the expression of integrinαvβ6.The control cells were those that transfected with the negative-siRNA or those cultured only with liposome-2000.Then,MTT assay was used for detecting the changes of survival and proliferation of AGS cells;adhesion assay for the ability of AGS adhering on the extracellular matrix(ECM);zymograph assay for the secretion of MMP-9;transwell-cabin assay for the infiltration of AGS cells;subcutaneous implantation in athymic mouse for the tumour formation. The role of integrinαvβ6 was analysised via comparing the difference between the test data of treated cells and the control cells.Results 48h later after the transfection of siRNA,the results of RT-PCR and Western blotting indicated that the expression of integrinαvβ6 were inhibited both in mRNA and protein levels.MTT assay found that the survival ability of AGS cells was only 49.8%of the control cells(P＜0.01);adhesion assay found the ability of AGS adhering to the ECM was only 79%of the contorl cells (P＜0.01);zymography assay found the ability of MMP-9 secretion inαvβ6-siRNA transfected cells decreased markedly(P＜0.01 vs the contorl); transwell-cabin assay indicated that the infiltration of the cells transfected withαvβ6-siRNA weakened obviously(P＜0.01 vs the contorl);tumour formation assay found that the time for the macroscopic tumour was about 6 days after the subcutanious implatation in the control cells,while it was 13 days in theαvβ6-siRNA transfected cells(P＜0.01 vs the contorl);and 6 weeks later after the subcutanious implatation,the average volume of the tumor formed from the control cells was about 820mm~3,while it was about 160 mm~3 in theαvβ6-siRNA transfected ceils(P＜0.01 vs the contorl).Conclusions Integrinαvβ6 has an important role in promoting survival,proliferation, infiltration and metastasis of gastric carcinoma AGS cells,and inhibiting the expression of integrinαvβ6 could inhibite the progression of gastric carcinoma.Significance We revealed the important role of integrinαvβ6 in promoting survival, proliferation,infiltration and metastasis of gastric carcinoma AGS cells, enriched the knowledge of integrinαvβ6,and provided the exact evidence for the targeted therapy onαvβ6 for gastric carcinoma. Part Two The role of integrinαvβ6 in the apoptosis of gastric carcinoma and colon cancer cellsObjective To study the role of integrinαvβ6 in the apoptosis of gastric carcinoma and colon cancer cells.Methods After inhibiting the expression of integrinαvβ6 withαvβ6-siRNA in AGS cells, or blocking the function of integrinαvβ6 with the monoclone antibody 10D5 in HT29 cells,we used Hoechst dyeing to detect the changes of morphology and the rate of apoptosis of the cells;caspase-3 activity assay for the changes of the activity of caspase-3;Western blotting for the expressoins of the apoptosis related proteins such as Bcl-2,Bax,Fas and Fas ligand;and for the changes of the distribution of cytochrome C and the expression of phosphorylated ERK, p38 and JNK.Results After inhibiting the expression of integrinαvβ6 withαvβ6-siRNA in AGS cells, or blocking the function of integrinαvβ6 with 10D5 in HT29 cells,AGS and HT29 cells all presented apoptotic morphological changes,such as the blue emission light in apoptotic cells was much bright than the control cells, condensed chromatin could be found in lots of the treated cells and some of them formed the structure of apoptotic bodies,which is one of the classic characteristics of apoptotic cells.The apoptotic rate of theαvβ6-siRNA transfected AGS cells and the 10D5 blocked HT29 cells all increased markedly (P＜0.01 vs the control).Caspase-3 activity assay found that the activity of caspase-3 increased obviously both in the treated AGS and HT29 cells(P＜0.01 vs the contorl);Western blotting indicated that in theαvβ6-siRNA transfected AGS cells,the expression of Bcl-2 decreased,while those of Bax,Fas and FasL increased(P＜0.01 vs the contorl);and cytochrome C released from mitochondrion into cytoplasma.The expression of phosphorylated ERK decreased,and phosphorylated p38 increased(P＜0.01 vs the contorl),while that of phosphorylated JNK did not change(P＞0.05 vs the contorl).However, in the 10D5 blocked HT29 cells,the expression of Bcl-2 increased together with that of Bax decreased(P＜0.01 vs the contorl),while those of Fas and FasL had no change(P＞0.05 vs the contorl).The cytochrome C released from mitochondrion into cytoplasma.The expression of P-ERK was downregulated (P＜0.01 vs the contorl),while those of P-p38 and P-JNK had no change (P＞0.05 vs the contorl).Conclusions Integrinαvβ6 has an important role in inhibiting apoptosis both in gastric carcinoma AGS cells and colon cancer HT29 cells,and knockdown of the expression or blocking the function of integrinαvβ6 both could induce apoptosis of the cells.Death receptor pathway and mitochondric pathway are involved in the apoptotic process of AGS cells,but only mitochondric pathway play a part in the apoptosis of HT29 ceils.Meanwhile,MAPK signal patyway participates in this process,and P-ERK plays an anti-apoptosis role both in AGS and HT29 cells,while P-p38 plays a pro-apoptosis role only in AGS cells.Significance We revealed the important role and the intimate mechanisms of integrinαvβ6 in inhibiting apoptosis of gastric and colon cancer cells,enriched the knowledge of integrinαvβ6,and provided the exact evidence for the targeted therapy onαvβ6 for gastric carcinoma.Part Three The role of integrinαvβ6 in the prognosis of gastric carcinomaObjective This study was to investigate the relationships between integrinαvβ6 expression and the clinical- pathological features of gastric carcinoma and whether integrinαvβ36 can act as a prognostic indicator in gastric carcinoma.Methods We generated the microarray of 300 human gastric carcinoma specimens,and used the method of immunohistochemistry to investigate the expression ofαvβ6 in them and the relationships between the expression ofαvβ6 and the clinical-pathological features of the tumors.Meanwhile,we retrospectively analyzed the relationship betweenαvβ6 expression and the survival times of the patients.Results The expression ofαvβ6 was detected in 36.7%of gastric carcinomas,and the expression was associated with Lauren type,differentiation,N stage and TNM stage of the tumors(the P values were 0.004,0.035,0.024 and 0.001 respectively).The Kaplan-Meier plot showed that patients withαvβ6 negative had much longer survival times than those withαvβ6 positive(P＜0.0001).The survival estimates showed a striking difference in median survival between the negative and positiveαvβ6 expression patients,especially in early stage tumors. Univariate analysis indicated that significant factors for prognosis includedαvβ6 expression,differentiation,TNM stage,T stage,N stage,M stage and R classification(R0:potentially curative resection;R1:had residual microscopic disease after resection;R2:had residual macroscopic disease after resection), while in multivariate analysis using Cox regressing model,onlyαvβ6 expression,M stage,TNM stage and R classification retained significant for prognosis..Conclusions Positiveαvβ6 expression in gastric carcinoma is linked to significantly reduced survival times,and what even more important is that its value as a prognostic marker is significant for early-stage tumors.Significance We revealed the relationships between integrinαvβ6 expression and the clinical- pathological features of gastric carcinoma,confirmed the important prognostic role ofαvβ6 in gastric carcinoma for the first time,enriched the knowledge of integrinαvβ6,and provided the exact evidence for the targeted therapy onαvβ6 for gastric carcinoma.